BRPI0610958A2 - method for producing a bioengineered form of tissue plasminogen activator - Google Patents

Abstract

The present invention relates to the recombinant method used for producing a soluble form of a tissue piasminogen activator variant. In this embodiment, threonine at position 103 of the endogenous tissue plasminogen activator is replaced by an asparagine, leading to a new glycosylation site. At position 117 of the tissue piasminogen activator, asparagine was replaced by glutamine, leading to the removal of an N-linked glycosylation site. At position 296-299, the amino acids plant, histidine, arginine, and arginine were replaced by four alanine amino acids. . The invention further relates to the unusual synthesis of the nucleic acid sequence encoding the tissue plasminogen activator, transformation of nucleic acid sequences constructed into competent bacteria and their subcloning into mammalian expression vectors for expression of the desired protein. DNA constructs that comprise the control elements associated with the laresse gene have been described. The recombinant human tissue plasminogen activator according to the invention, and its salts and functional derivatives, may comprise the active ingredient of pharmaceutical compositions for the treatment of myocardial infarction and cerebrovascular acid in patients. These compositions are yet another aspect of the present invention.

Description

Report of the Invention Patent for "METHOD TO PRODUCE A FORM OF BIOENGENHARIA TISSUE PLASMINOGEN DACTIVATOR".

Technical Field of the Invention

The present invention relates to the recombinant method used to produce a soluble form of a human tissue plasminogen activator variant. In this embodiment, threonine at position 103 of the endogenous tissue plasminogen activator is replaced by an asparagine, leading to a new glycosylation site. At position 117 of the tissue deplasminogen activator, asparagine was replaced by glutamine, leading to the removal of an N-linked glycosylation site. At position 296-299, the amino acids lysine, histidine, arginine, and arginine were replaced by four alanine amino acids.

The invention further relates to the unusual synthesis of the nucleic acid sequence encoding the tissue plasminogen activator, transformation of nucleic acid sequences constructed into competent bacteria, and subcloning thereof into mammalian expression vectors for expression. of the desired protein.

DNA constructs comprising the control elements associated with the gene of interest have been described.

The recombinant human tissue plasminogen activator according to the invention, and its salts and functional derivatives, may comprise the active ingredient of pharmaceutical compositions for the treatment of myocardial infarction and stroke in patients. These compositions are yet another aspect of the present invention.

Background of the Invention

Plasminogen activators are enzymes that activate zymogen oplasminogen to generate serine proteinase plasmin, which degrades fibrin. Among the plasminogen activators studied are streptokinase, urokinase and tissue plasminogen activator (t-PA).

The mechanism of action of each of these plasminogen activators differs. Streptokinase forms a complex with plasminogen, generating plasmin activity, urokinase cleaves plasminogen directly, and t-PA forms a ternary complex with fibrin and plasminogen, leading to activation of plasminogen at the clot site.

Tissue-like plasminogen activator (t-PA), a multi-domain glycosylated serinaprotease, is a fibrin-specific plasminogen activator and a very effective thrombolytic agent. t-PA is a recombinant protein whose main application is in the treatment of patients with myocardial infarction and stroke. It was first characterized in 1979 as an important and potent biological pharmaceutical agent in the treatment of various vascular diseases due to its high fibrin specificity and potent ability to dissolve blood clots in vivo.

Natural t-PA has a plasma half-life of about its minutes or less. Due to its rapid clearance of circulation, t-PA must be infused to achieve thrombolysis. Administration of dosages with early loading and increased t-PA concentrations demonstrated faster and more complete lysis compared to the usual infusion protocol and early potency correlates with better survival rate. Massive administration could further improve the rate of lysis by rapidly exposing the target clot to a higher enzyme concentration, but a single massive administration of natural or wild-type t-PA cannot generally be used due to its clearance rate. .

Many researchers have produced longer half-life versions of t-PA that could be massively administered, but all variants have had significantly decreased fibrinolytic activities.

Accordingly, it is an object of the present invention to provide a recombinant method used for the production of a molecule with reduced purification rate while retaining complete fibrinolytic activity, and systematic mutagenesis studies have been applied to t-PA in its various domains. Such a drug would also have a higher specificity with higher affinity for a recent thrombus and would produce less circulating plasmin. Consequently, the incidence of intracerebral hemorrhage (ICH) and other episodes of non-cerebral bleeding would be lower. The drug resistance to PAI-1 would also be productive.

Summary of the Invention

The present invention relates to the recombinant method used to produce a soluble form of a human tissue plasminogen activator variant. In this embodiment, threonine at position 103 of the endogenous tissue plasminogen activator is replaced by an asparagine, leading to a new glycosylation site. At position 117 of the tissue deplasminogen activator, asparagine was replaced by glutamine, leading to the removal of an N-linked glycosylation site. At position 296-299, the amino acids lysine, histidine, arginine, and arginine were replaced by four alanine amino acids.

A specific aspect of the invention relates to the novel nucleic acid sequence synthesis encoding the plasminogen activator, transformation of the competent embacteria constructed nucleic acid sequences and subcloning them into mammalian expression vectors for expression of the desired protein.

Yet another aspect of the invention provides vital biologically functional circular plasmid DNA vectors incorporating the DNA sequences of the invention and stably transformed or transfected host organisms with said vectors.

The invention accordingly provides novel methods for the production of useful polypeptides comprising the cultured development of such transformed host cells, particularly mammalian cells, under conditions that facilitate the large-scale expression of vector-borne exogenous DNA sequences and the isolation of desired polypeptides from the medium. growth, cell lysates or cell membrane fractions.

Detailed Description of Figures and Sequences

Figure 1 illustrates the paired alignment of non-optimized and codon optimized version sequence sequences of the DNA nucleotide coding encoding the tissue plasminogen activator;

Figure 2 illustrates the sequence alignment of unusually synthesized TE-NECT cDNA (TNK-tPA_synthetic) with the established sequence of the TNK-tPA gene;

Figure 3 illustrates the sequence alignment of the unusually synthesized TE-NECT-Opt cDNA (TNK-tPA-Opt_synthetic) with the established sequence of the TNK-tPA-Opt gene;

Figure 4 shows the restriction digested and gel purified TENECT, TEECT-Opt epcDNA3.1 DA / 5-His fragments;

Figure 5 illustrates restriction digestion analysis of pcDNA3.1-TENECT DA / 5-His / TNK-tPA and pcDN3.1-TENECT-OptA / 5-His / TNK-tPA-Opt clones;

Figure 6 illustrates restriction digestion analysis of clonesPcDNA3.1-TENECT / V5-His / TNK-tPA and PcDNA3.1-TENECT-OptA / 5-His / TNK-tPA-Opt using enzymes that cleave cDNAs from TENECT and TE-NECT-Opt internally;

Figure 7 illustrates the Construction Map: PcDNA3.1-TENECTA / 5-His / TNA-tPA;

Figure 8 illustrates the Construction Map: PcDNA3.1 -TENECT-Opt / V5-His / TNK-tPA-Opt;

SEQ ID Ne: 1 is the nucleotide sequence encoding the recombinant tissue plasminogen activator;

SEQ ID N9: 2 is the codon optimized version of the denucleotide sequence encoding the recombinant tissue plasminogen activator.

Detailed Description of the Invention

Several methods have been described for the expression of recombinant proteins in higher eukaryotic systems. CHO-K1, HEK-293 (and variant) expression systems have now been established as the predominant systems of choice for mammalian protein expression. Improvements in vector construction, selection of selectable pathways, and advances in gene yield and high throughput screening strategies have made it relatively common to establish recombinant cell lines with high specific yields and have reduced the time required to develop them. development of cell lines. Recent advances in expression technologies using traditional viral promoter-based expression vectors include the development and enhancement of biscistronic expression strategies using sequences of inter-ribosome (IRES) entry sites or alternative junctions.

Example 1

The DNA sequences encoding the tissue plasminogen activator have been synthesized by an innovative approach. This approach allows for better codon optimization with respect to the mammalian specific cell line being used. In addition, synthetic DNA has become the subject of eukaryotic / prokaryotic expression which provides isolable amounts of polypeptides which exhibit naturally occurring t-PA biological properties as well as in vivo and invitro t-PA biological activities.

The nucleotide sequence encoding the recombinant tissue plasminogen activator (TENECT 1) was represented in SEQ ID NQ:

1. Codons in the tissue plasminogen activator coding DNA sequence, which have been altered as part of the codon optimization process to ensure optimal expression of recombinant proteins in mammalian cell lines such as CHO K1 and HEK 293, have been highlighted in capital letters. SEQ ID N9: 2 represents the codon-optimized denucleotide sequence encoding the tissue plasminogen activator (TENECT 2).

Paired alignment of codon-optimized non-optimized denucleotide sequences encoding tissue plasminogen activator was depicted in Figure 1.Example 2

Verification of authenticity of unusually synthesized cDNA encoding tissue plasminogen activator

Verification of the authenticity of unusually synthesized cDNA molecules, as supplied by the supplier, was performed by automated DNA sequencing, and the results are shown in Figures 2 and 3.

Example 3

Subcloning of TENECT and TENECT-Opt cDNAs into mammalian cell-specific expression vector pcDNA3.1DA / 5-His

After verifying the authenticity of the innovatively synthesized DNA molecules (TENECT and TENECT-Opt) by automated DNA sequencing as indicated above, TENECT and TENECT-Opt were individually subcloned into the pcDNA3.1D mammalian cell-specific expression vector. / V5-His to generate the transfection ready constructs. The details of the procedures are as follows:

A. Reagents and Enzymes:

1. QIAGEN gel extraction kit and PCR purification kit;

2._DNA of the pcDNA3.1 D / V5-His vector (Invitrogen)

<table> table see original document page 7 </column> </row> <table>

All reactions were conducted as recommended by the manufacturer. For each reaction, the 10x reaction buffer was diluted to a final concentration of 1 x.

B. Restricted vector and insert digestion

- Procedure:

The following DNA and restriction enzyme samples were used: <table> table see original document page 8 </column> </row> <table>

- Restriction enzyme digestion reactions:

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The reaction was mixed, centrifuged and incubated for two hours at 37 ° C. Restricted digestion was analyzed by aga-rose gel electrophoresis. The expected digestion pattern was observed with characteristics of a gene fragment cut-off of ~ 1,700 base pairs (for Rxn n— 3 and 4) and a main supper fragment of the vector with -5,5 kb for the vector (Rxn n— 1 e2) was observed. The 1,700 bp DNA fragments, representing the TENECT and TENECT-Opt DNac's, were purified separately by the gel extraction method using the gelQIAGEN extraction kit. The main strand of the digested -5.5 kb vector from the mammalian expression vector, pcDNA3.1D / V5-His, was also purified using the same. Following restricted digestion and gel extraction of DMAc and vector DNA fragments, an aliquot (1-2 microliters) of each purified DNA sample was analyzed using agarose gel electrophoresis to verify purity and integrity as illustrated in Figure 4 below:

C. Binding of pcDNA3.1D / V5-His Main Chain with TENECT and TENECT-OptDNAs The concentration of the digested and purified vector DNA and insert fragments was estimated (see Figure 4 above) and binding was established. as follows:

<table> table see original document page 9 </column> </row> <table>

Reactions were gently mixed, centrifuged and incubated at room temperature for 2-3 h. Competent DH10 cells were transformed with the contents of the ligation reaction mixtures.

D. Restriction digestion analysis of putative clones of pcD-NA3.1-TENECT / V5-His / TNK-tPA and pcDNA3.1D-TENECT-Opt / V5-His / TNK-tPA-Opt

Plasmid DNA was individually purified from the colonies obtained on ampicillin-containing L.B agar plates, and the presence of the desired cDNA insert was confirmed by restriction digestion analysis of the isolated plasmid DNA, as illustrated in Figure 5.

According to results obtained after restriction digestion of several putative clones containing pcDNA3.1-TENECT / V5-His / TNK-tPA and pcDNA3.1-TENECT-Opt / V5-His / TNK-tPA-Opt, some of the clones which exhibited the desired restriction pattern were selected for further analysis of restriction restriction digestion using restriction enzymes that cleave TENECT and TENECT-Opt cDNAs internally to fragment fragments of varying sizes, as illustrated in Figure 6.

Most pcDNA3.1-TENECT / V5-His / TNK-tPA clones epcDNA3.1-TENECT-OptA / 5-His / TNK-tPA-Opt selected for restriction mapping analysis produced the expected fragment sizes based on the occurrence of known internal restriction sites, and thus these clones will be further verified by DNA sequencing analysis.

Maps of recombinant expression constructs made using the unusually synthesized TENECT and TENECT-Opt cDNAs are pictorially shown in Figures 7 and 8.

Example 4

Maintenance and Propagation of Human t-PA Construction

The maintenance and propagation of the human cDNA-encoding cDNA construct will be done in usual bacterial cultures. Glycerin stocks of all clones would be kept and stored at -70 ° C.

Example 5

Transient and Stable Expression of Recombinant Proteins in CHO-K1 Cells

Transient and stable expression of human t-PA was done using Chinese hamster ovary (CHO) cells, a mammalian cell line that is FDA approved to produce therapeutic proteins. Transient expression is useful for verifying expression of a construct and for rapidly obtaining small amounts of a recombinant protein.

Protein expression would be further analyzed using analytical tools such as Western blot and functional assays.

Example 6

Purification of Recombinant Tissue Plasminogen Activator

After the establishment of a contaminant-free cell line, as directed by regulatory agencies, that overexpresses the desired recombinant protein, purification strategies will aim for process economics, speed to market, high production capacity, reproducibility, and maximum product purity with functional stability and structural integrity as the most important objectives. For this purpose, a combined approach with filtration (normal and tangential flow filtration) and chromatography should be explored. Process qualification requirements and acceptance criteria studies will be conducted in 3 batches. Accordingly, the present invention considers the following steps in the purification process and / or usual methods known per se:

The. Clarification and initial concentration of crude culture broth using normal and tangential flow filtration procedures;

B. Ultrafiltration / Dialysis Filtration (based on tangential flow filtration);

ç. Chromatographic Step I: Affinity chromatography using heparin, lysine, metal chelate (zinc) in Sepharose, and immobilized Mabs on Sepharose. More preferably, lysine in Sepharose will be used in downstream unit operations;

and. Chromatographic Stage II: anion exchange chromatography using DEAE-cellulose;

f. Virus removal and sterile filtration;

g. Endotoxin removal.

Note: In addition an anion exchangers-based flow such as cellufine sulfate will be used for the selective binding of process contaminants, endogenous / adventitious viruses and extractable fractions of the column.

Example 7

Target Protein Identity Establishment Using Biochemical, Immunological, and Physical Chemical Methods

Percent recovery of total protein at each stage will be quantified using the bicinoninic acid (BCA) procedure and the Bradford dye binding method. Target protein concentration will be routinely determined at each stage of purification, using highly specific and reliable enzyme-based assays, such as ELISA capture, using standard sequence t-PA polyclonal / monoclonal antibodies. Qualitative and target-specific Western analysis will be followed at each stage. Reverse phase chromatography, isoelectric focusing and two-dimensional gel electrophoresis will be employed to evaluate the purified product. Secondary structural analysis would be performed using extreme UV circular dichroism. Molecular mass and oligomeric status will be investigated using eMALDI-TOF size exclusion. Research will also focus on protein stability in relation to pH and temperature. <110> ftvestha Gengraine Technologies .í? * T Ltd.Môíawala Pátell, Villoo

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Glu Ala Hís Goes Arg Leuyr Pro Ser. Ser Arg Cys Thr Ser Gln His465 470 475 • • '4B0

ttg ctg aac cgc act gtt acc gac aat atg ctg tgt gcc ggt gat acc1488

Let * Read Ãsh Arg Thr Go Ebr Asp Asn K «t L @ u Cys Ma Gly Asp Thr4B5 490 495

agg tco ggg ggc cct cag gcc aat ctg cat gac gcg tgc cag ggg gac1536

Arg Ser Gly Gly Pro / filn Agn Wing! «Ew Mts Asp Cys Wing Gln Gly Asp500 505 510

tec ggc ggg cec ctg gtg tgt ttg aac gat gga agg atg acc ctg gtc1584

Be Gly Gly for Leu Go Cys I ^ i Asti Asp Gly Arg Mefc ITir Leu Go515 520 525

till tet tgg-ggc ctg ggc tgc ggc cag aag gat gtg cca ggc1632

Gly lis lie Ser 1? Rp Gly Leu Gly Cys Gly Gln Lys Asp Goes Exo © iy530 535 540

gtc tac acc aag gtg acg aac tac ctg gac

Val T.yxr Thr Lys Will Thr Asn 5? Yr Read Asp Trp lie Arg Asp Asn Met

545 550 555 560

agg cec tga

1683Arg Pro

Claims (9)

Process for the preparation of a biologically active Tissue Plasminogen Activator in vivo, comprising the steps of: (a) developing, under appropriate nutrient conditions, transformed or transfected host cells with an isolated DNA sequence selected from the group consisting of ( (i) DNA sequences disclosed in SEQ ID N9: 1 and SEQ ID N9: 2, or (ii) DNA sequences that hybridize under severe conditions to the DNA sequences defined in (i) and (ii) or their filaments complementary; and (b) isolating said recombinant Tissue Plasminogen Activator product from them. 2. A process for preparing a biologically active Tissue Plasminogen Activator product in vivo, comprising the steps of transforming a host cell with a synthesized DNA sequence represented in SEQ ID NQ: 1 or 2, encoding Tissue Plasminogen Activator, and isolating said product of said host cell or growth rate thereof. A process according to claim 1 or 2, wherein said host cells are mammalian cells. A process according to claim 1 or 2, wherein said host cells are preferably CHO K1 cells. A process for producing a soluble form of Tissue Plasminogen Activator, which has the in vivo biological property of treating myocardial infarction and stroke, comprising the steps of: (a) developing, under appropriate nutrient conditions, mammalian cells comprising DNA promoter other than tissue plasminogen activating DNA promoter operably linked to DNA encoding the mature erythropoietin amino acid sequence of SEQ ID N9: -3; and (b) isolating said glycosylated erythropoietin polypeptide expressed by said cells. A process according to claim 5 wherein said promoter DNA is a viral promoter DNA. A process for preparing a biologically active tissue plasminogen activator product in vivo, comprising the steps of transforming a host cell with a vetorda construct Figure 7 or 8 and isolating said Teci-dual Plasminogen Activator product from said host cell. or the middle of its growth. The process of claim 7, wherein said vector is a mammalian cell specific expression vector and more preferably the vector depicted in Figures 7 and 8. A pharmaceutical composition comprising a therapeutically effective amount of Tissue Plasminogen Activator and a pharmaceutically acceptable diluent, adjuvant or carrier, wherein said Tissue Plasminogen Activator is purified from cultured mammalian cells.